Genetic engineering in plants

Genetic engineering or recombinant DNA (rDNA) technology involves artificial transfer of

genes or gene fragments from one organism to another to produce novel traits in the
recipient living organism.

The development of recombinant DNA technology (rDNA technology) permitting the

transfer of genetic material between widely divergent species has opened a new era of
research into the structure and function of the genome. The rDNA technology is defined
as "the formation of new combinations of heritable material by the insertion of nucleic
acid molecules, produced by whatever means outside the cell, into any virus, bacterial
plasmid or other vector system so as to allow their incorporation into a host organism in
which they do not naturally occur but in which they are capable of continued propagation.
The rDNA technology has provided the means to achieve:
1) The fractionation of individual DNA components of complex genomes
2) The amplification of cloned genes
3) The opportunity to study the expression of individual genes thus cloned and
4) The potential to create new genetic combinations

There are several other terms that can be used to describe the technology, including
gene manipulation, gene cloning, genetic modification and genetic engineering.
The term genetic engineering is often thought to be rather emotive or trivial, yet it is
probably the label that most people would recognize.

Any rDNA experiment has four essential steps:

1) Generating DNA fragments
2) Cutting and joining the DNA fragments to vector DNA molecules
3) Introducing the vectors carrying the foreign DNA into host cells where they can
replicate and
4) Selecting the clone(s) of recipient cells that have acquired the recombinant DNA
molecules
 Steps involved in rDNA technology

Generating DNA fragments

One of the most important problems prior to rDNA experiment is to separate the DNA
fragments from the total genomic DNA. This is normally accomplished either by
fragmentation of DNA or synthesis of new DNA molecule. The fragmentation of DNA
molecule can be achieved by mechanical shearing. The long thin threads which
constitute duplex DNA molecules are sufficiently rigid to be very easily broken by shear
forces in solution. In this method, high molecular weight DNA is sheared to population of
molecules with a mean size of about 8kb pairs by stirring at 1500 rpm for 30 minutes.
Breakage occurs essentially at random with respect to DNA sequence producing termini
consisting of short single stranded regions which may be repaired later. The other
sophisticated technique available to generate DNA fragments involves using restriction
endonucleases about which discussion is made in the subsequent section. Other two
possible sources for generating DNA fragments for cloning are complementary DNA
(cDNA) synthesis using mRNA as a template and artificial synthesis of DNA molecule.

cDNA synthesis
Fundamental differences exist between the genomes of prokaryotes and eukaryotes. In
prokaryotes, the coding sequences (exons) are not intervened by non-coding sequences
(introns) whereas in eukaryotes the genes are generally split; the coding regions are
interspersed with non coding DNA. This makes the expression of eukaryotic genes in
prokaryotes a tough task.

Synthesis of cDNA from mRNA

To overcome this problem, cDNA synthesis or artificial DNA synthesis can be well
exploited. In cDNA synthesis, the eukaryotic mRNA is used as a template to generate
DNA. This can be achieved by making a complementary copy of the mRNA using the
enzyme reverse transcriptase and whose function is to synthesize DNA upon an RNA
template. At first, the enzyme was called RNA dependent DNA polymerase.

A DNA copy is made by hybridizing oligo-T primers, 10 to 20 nucleotides in length, to the

3' end of purified mRNA. Avian myeloblastosis virus (AMV) reverse transcriptase is used
to synthesis a cDNA copy of the primed molecule. In the resultant RNA-DNA hybrid, the
RNA can be destroyed by alkaline hydrolysis to which DNA is resistant. Thus, a single
stranded cDNA is obtained which can be converted into a double stranded form in
second DNA polymerase reaction. In the 3' end of the cDNA self-complementary occurs
thus producing a hair-pin or snap-back structure. This acts as a primer for duplex DNA
synthesis by DNA polymerase. The hair-pin loop is trimmed away by treatment with
single strand specific nuclease SI, giving rise to a fully duplex molecule. The power of
this technique is that only a fraction of the genome (that fraction which is transcribed into
mRNA) is copied. The resulting cDNA clones can be subsequently be used as probes to
identify genomic fragments contained in a genomic library.
Chemical synthesis of DNA
Although the methods for generating DNA fragments mentioned above are those most
commonly used, the chemical synthesis is considered as an increasingly important
method for generating DNA molecules. The chemical synthesis of specific gene
sequences, regulatory sequences, oligonucleotide probes, primers and linkers is a
technique in which solid phase synthesis is adopted. In chemical synthesis of DNA, two
important strategies adopted are described below.

Phosphodiester method
In the phosphodiester method, 3’ and 5’ hydroxyl groups of deoxyribose are protected
(R1 and R2). In this method, the phosphorus group between the two nucleosides is
unprotected. These compounds are therefore soluble in organic solvents to a limited
extent. The first significant successes, such as the synthesis of the genes for alanine
and tyrosine suppressor tRNA for yeast and E. coli respectively were gained with the
phosphodiester method.

Phosphotriester method
The phosphotriester method for the synthesis of oilgodeoxyribonucleotides proceeds
essentially in two steps: 1) preparation of suitably protected monomers and 2) coupling of
the monomers in the desired sequence by an appropriate phosphorylation procedure.

In both protocols the 3’ and 5’ hydroxyl groups of the deoxyribose sugar are suitably
protected (R1 and R2). In the phosphotriester method a third protecting group (R3) is
used for the hydroxyl group at the inter nucleotide bond. Chemical synthesis of DNA has
found an extraordinary number of applications in gene technology which include
synthesis of partial or total gene sequences, primers for DNA and RNA sequencing,
hybridization probes for the screening of RNA, DNA and cDNA or genomic libraries and
adapters and linkers for gene cloning
Cutting and joining the DNA fragments to vector DNA molecules
Restriction endonucleases: Tool for cutting DNA molecules
Techniques for cutting of DNA molecules into discrete fragments by specific enzymes
were virtually unknown until the late sixties. A solution to this fundamental problem
eventually grew from long standing research into the phenomenon of host controlled
restriction and modification system.

Host controlled restriction and modification phenomenon can be well explained with the
following example. If a stock preparation of phage is allowed to grow upon E. coli strain
C and this stock is then tried upon E. coli C and E. coli K, the titres observed on these
two strains will differ by several orders of magnitude, the titre on E. coli K being the
lowest. The phages are said to be restricted by the second host strain (E.coli K) and the
phenomenon is called restriction. When those phage that do result from the infection of
E. coli K are now replated on E. coli K they are no longer restricted; but if they are first
cycled through E. coli C they are once again restricted when plated upon E. coli K. The
non-heritable change conferred upon the phage by the second host strain (E. coli K) that
allows it to be replicated on that strain without further restriction is called modification.
These processes can occur whenever DNA is transferred from one bacterial strain to
another. Conjugation, transduction, transformation and transfection are all subject to the
constraint of host controlled restriction and this process is made possible by the
enzymes called restriction endonucleases.
Ligases: Tool for joining DNA fragments
Joining DNA fragments of various types is yet another fundamental step in rDNA
technology. This process is otherwise called as ligation and is achieved by the catalytic
reaction of enzymes called ligases. These enzymes catalyses the formation of
phosphodiester bonds between DNA molecules. The ligase enzymes of E. coli and
phage T4 have the ability to seal the single stranded nicks between nucleotides in a
duplex DNA.

Ligation
Although the reactions catalyzed by the enzymes of E. coli and T4 infected E. coli are
similar, they differ in their cofactor requirements. The T4 enzyme requires ATP, while the
E. coli enzymes require NAD+. In each case the cofactor is split to form an enzyme-AMP
complex. The complex binds to the nick, which must expose a 5'- phosphate and 3'-OH
group, and makes a covalent bond in the phosphodiester chain.

The other enzyme having utility in ligation is terminal deoxynucleotidyl- transferase. This
adds an entire nucleotide to 3’ end of the chain. It requires a source of energized
nucleotides and simply adds them to the growing chain. This means that, if some DNA is
mixed with terminal transferase and just one nucleotide, say the adenine nucleotide, the
chain will grow as succession of adenines at the 3’ end of the strand. If another chain is
incubated with terminal tranferase and thymine nucleotides it will have a protruding
strand that is all thymine. If the above two strands are mixed together the
complementary base pairing between the protruding strands will give duplex DNA.

Prevention of self-ligation
In rDNA technology, prevention of self-ligation in vector DNA molecules or passenger
DNA molecules is considered more important. Generally vector DNA molecules are
highly susceptible to self ligation thus forming recircularised DNA molecules. The
presence of self ligated molecules reduces the probability of recovering desired
recombinant clones. Self ligation can be reduced to some extent by adopting homo
polymer tailing. Wherever homopolymer tailing is undesirable other strategies like
directional cloning and dephosphorylation of termini can be followed.

Directional cloning: Directional cloning is otherwise called forced cloning. This is

possible in a vector having two or more target sites in a non essential portion of the
DNA. Cleavage at these sites cause the removal of non essential DNA and produce a
vector molecule with two different termini which are not complementary so that the
individual vectors cannot recircularise.

Dephosphorylation of termini: The main function of DNA ligase is to produce a

phosphodiester bond between adjacent nucleotides if one contains a 5' PO4 group and
the other a 3' –OH group. Thus, removal of terminal 5' PO4 groups from the cleaved
DNA will prevent self ligation. Dephosphorylation of termini can be carried out by treating
linearised DNA with bacterial alkaline phosphatase. The dephosphorylated DNA
molecules can be religated with phosphorylated passenger DNA to produce functional
recombinant DNA molecules.

rDNA techniques for the production of transgenic

Genetic engineering or recombinant DNA (rDNA) technology involves artificial transfer of

genes or gene fragments from one organism to another to produce novel traits in the
recipient living organism. The important tools used in rDNA technology include:

Enzymes for DNA manipulation

The first step in the construction of a recombinant DNA molecule, involves cleaving DNA
molecules at specific points and recombining them together again in a controlled
manner. The two main types of enzymes commonly used for this purpose are restriction
endonucleases and DNA ligases. These enzymes form the backbone of rDNA
technology. Restriction endonucleases cut DNA into defined fragments by targeting
junction of specific sequences of the genetic coding and DNA ligases recombine them
by consolidating loose bonds for creating large fragments. These enzymes are very
specific in their action.

Vectors
The function of the vector is to enable the foreign genes to get introduced into and
become established within the host cell. Naturally occurring DNA molecules that satisfy
the basic requirements for a vector are plasmids and the genomes of bacteriophages
and eukaryotic viruses. They are further classified as cloning and expression vectors
depending on the stage of genetic engineering at which these vectors are used.
Expression hosts
The functional cell into which the composite DNA molecule carrying the required gene
needs to be introduced is called the expression host. The choice of the best host-vector
system for the expression and large-scale production of a particular protein is based on
considerations of the complexity of the protein to be expressed and the yield and
quantities needed.

Marker genes
Marker genes and reporter genes are utilized for selection and identification of the
clones. These use phenotypic markers, identification from a gene library and DNA
sequencing. DNA sequencing helps in determining the precise order of nucleotides in a
piece of DNA.

Construction and Identification of recombinant DNA molecules

Recombinant DNA (rDNA) has various definitions, ranging from very simple to strangely
complex. The following are three examples of how recombinant DNA is defined:
1. A DNA molecule containing DNA originating from two or more sources.
2. DNA that has been artificially created. It is DNA from two or more sources
that is incorporated into a single recombinant molecule.
3. According to the NIH guidelines, recombinant DNA are molecules
constructed outside of living cells by joining natural or synthetic DNA
segments to DNA molecules that can replicate in a living cell, or molecules
that result from their replication.

Description of rDNA
Recombinant DNA, also known as in vitro recombination, is a technique involved in
creating and purifying desired genes. Molecular cloning (i.e. gene cloning) involves
creating recombinant DNA and introducing it into a host cell to be replicated. One of the
basic strategies of molecular cloning is to move desired genes from a large, complex
genome to a small, simple one. The process of in vitro recombination makes it possible
to cut different strands of DNA, in vitro (outside the cell), with a restriction enzyme and
join the DNA molecules together via complementary base pairing.

Techniques
Some of the molecular biology techniques utilized during recombinant DNA include:
 1. The study and/or modification of gene expression patterns
Gene expression is the process by which a gene's coded information is
converted into the structures present and operating in the cell. Expressed
genes include those that are transcribed into mRNA (messenger RNA) and
then translated into protein, and those that are transcribed into tRNA (transfer
RNA) and rRNA (ribosomal RNA). Gene expression can be studied using
microarray analysis, which is a method of visualizing the patterns of gene
expression of thousands of genes using fluorescence or radioactive
hybridization.
2. Gene cloning: Gene cloning utilizing recombinant DNA technology is the
process of manipulating DNA to produce multiple copies of a single gene or
segment of DNA.
3. DNA sequencing
DNA sequencing is a lab technique used to determine the sequence of
nucleotide bases in a molecule of DNA.
4. Creation of transgenic plants and animals
A transgenic plant or animal is one who has been genetically engineered, and
usually contains genetic material from at least one unrelated organism, such
as from a virus, other plant, or other animal.

Processes
The following is a summary of the process of making recombinant DNA:
1. Treat the DNA taken from both sources with the same restriction
endonuclease.
2. The restriction enzyme cuts both molecules at the same site.
3. The ends of the cut have an overhanging piece of single-stranded DNA called
“sticky ends.”
4. These sticky ends are able to base pair with any DNA molecule that contains
the complementary sticky end.
5. Complementary sticky ends can pair with each other when mixed.
6. DNA ligase is used to covalently link the two strands into a molecule of
recombinant DNA.
7. In order to be useful, the recombinant DNA needs to be replicated many
times (i.e. cloned). Cloning can be done in vitro, via the Polymerase Chain
 Reaction (PCR), or in vivo (inside the cell) using unicellular prokaryotes (e.g.
E. coli), unicellular eukaryotes (e.g. yeast), or mammalian tissue culture cells.
Figure . A pictorial representation of the recombinant DNA process.

Generating DNA fragments using restriction endonucleases

Restriction endonucleases are enzymes that recognize specific sequences within duplex
DNA molecules and cut the DNA at or near these sites. More than 500 different
restriction endonucleases have been discovered. These enzymes can be grouped into
three types viz. Type I, II and III. For practical purposes, the Type I and III restriction
enzymes are not much used in rDNA technology. The real precision scissors are the
Type II enzymes. Type II restriction endonucleases recognize and cut DNA within
particular sequences of tetra, penta, hexa or hepta nucleotides which have an axis of
rotational symmetry. In the following examples, different restriction enzymes cut the DNA
at specific sequences as indicated by arrows.

Among the restriction enzymes, some enzymes cut the DNA molecules to give blunt
end fragments otherwise termed as flush end DNA fragments and some others produce
DNA molecules where one of the strands will have protruding 5' or 3' termini. These
fragments are called fragments with cohesive ends or sticky ends. The majority of the
recognition sequences for restriction endonucleases are palindromic, that is the
sequence is the same if read from 5' to 3' from both complementary strands.

The sites of cut made by endonucleases are called target sites or cleavage sites and
the number of these sites in a DNA molecule depends on the size of the DNA, its base
composition and the GC content of the recognition site. The number and size of the
fragments generated by a restriction enzyme depends on the frequency of occurrence of
the target site in the DNA to be cut. Assuming a DNA molecule with a 50 percent G+C
content and a random distribution of four bases, a restriction enzyme recognizing a
particular tetranucleotide sequence will be able to cut the DNA molecules into fragments
at once in every 44 (i.e. 256) nucleotide pairs. If the enzyme is having the property of
making cuts in hexanucleotide sequences means, the cuts will be made at every 46 (i.e.
4096) nucleotide pairs and an eight nucleotide recognition sequence 48 (65536) base
pairs.

Restriction enzymes that have the same recognition sequences can be isolated from
different bacterial species. Such enzymes are called isoschizomers. An example is
provided by Mbol (Moraxella boris) and Sau3A (Staph yhcoccus aureus), both of which
recognize the sequence GATC. Furthermore, some restriction enzymes generate
cohesive ends that can reanneal with identical termini produced by other enzymes. For
instance, DNA cleaved with BamHI (GGATCC) has compatible ends with DNA cleaved
with BglII, MboI, Sau3A, etc.

The number of restriction fragments made by an enzyme would be reduced if there is a

methylation of restriction sites. In some cases, the DNA recognition by an enzyme will
not be altered by methylation and enzymes of this nature are said to be enzymes with
star activity e.g. EcoRl, BarnHI and Sal1.

Ligation strategies
In rDNA technology, sealing discontinuities in the sugar-phosphate chains, otherwise
called as ligation, is vital step. This process is catalyzed by DNA ligase by repairing
broken phophodiester bonds. During ligation, the enzyme’s activity is influenced by
factors such as 1) substrate specificity, 2) temperature and 3) salt concentration
Ligation methods
Joining DNA fragments with cohesive ends by DNA ligase is a relatively efficient
process, which has been extensively used to create artificial recombinants. If the termini
of DNA fragments are not compatible, there are other methods to ligate the fragments.

Cohesive end ligation

The cohesive end ligation is possible when both the foreign DNA to be cloned and the
vector DNA possess the same molecular ends. The compatible sticky ends have been
generated by cleavage with the same enzyme on the same recognition sequences of
both foreign DNA and vector DNA. Using DNA ligase, these molecules can be ligated
without any problem.

Very often it is necessary to ligate DNA fragments with different and non-compatible ends,
or blunt ends with either staggered 3' or 5' ends. Incompatible DNA fragments with
recessed ends can be ligated by modifying their ends by any one of the following
methods viz., (i) filling in recessed 3' termini and (ii) renewal of 5' protruding termini.

Blunt end ligation

The E. coli DNA ligase will not catalyze blunt end ligation except under special reaction
conditions of macromolecular crowding. The unique property of T4 DNA ligase was used
to ligate DNA fragments with blunt ends involving short decameric oligonucleotides
called linkers.

Using linkers
Short oligonucleotides (decamers) which contain sites for one or more restriction
enzymes are used to facilitate the ligation process among the DNA fragments with blunt
ends.
 Joining of blunt end DNA to a vector using linkers

The linker molecules can be ligated to both ends of the foreign DNA to be cloned and
then treated with restriction endonuclease to produce sticky end fragments which can be
incorporated into a vector molecule that has been cut with the same restriction
endonuclease. Insertion by means of the linker creates restriction sites at each end of
the foreign DNA, and thus enables the foreign DNA excised and recovered after cloning
and amplification in the host bacterium.

Using adaptors
The other strategy adopted for ligating DNA fragments with blunt ends is using
adaptors. The adaptor molecules are synthetic deoxynucleotides that can be used to
join two incompatible cohesive ends, two blunt ends or a combination of both. Such
adaptors are of several types viz., preformed, conversion and single stranded
adaptors.

Preformed adaptors
Preformed adaptors are short DNA duplexes with at least one cohesive end. The
problem of internal cleavage of the insert DNA can be overcome by using a preformed
adaptor that will introduce a new restriction site. For example, an adaptor having BamHI
cohesive ends and sites HpaII and SmaI can be attached to passenger DNA and
inserted into a BamHI in vector. After cloning, passenger DNA can be excised from the
hybrid by using any one of the enzymes that recognize the restriction sites within the
adaptor region.

Use of preformed adaptors

Conversion adaptors
Conversion adaptors are synthetic oligonucleotides bearing different cohesive restriction
termini. Such adaptors enable vector molecules that have been cleaved with one
endonuclease to be joined to passenger fragments that have been cleaved with another.
Often these adaptors contain internal restriction sites that permit recovery of the
passenger fragment, for example, the EcoRI-BamHI adaptor contains a site for XhoI.

Use of conversion adaptor

 Single stranded adaptors
Single stranded adaptors can be used to make 3’-protruding cohesive ends compatible
with 5’ protruding ends. Such adaptors permit the insertion of passenger fragments into
sites on vectors from which they would otherwise be precluded because of incompatible
cohesive ends.

Homopolymer tailing
Homopolymer tailing is the other method adopted to clone blunt DNA molecules, especially
cDNA molecules.

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 Homopolymer tailing

The addition of several nucleotides of single type to the 3’ blunt end of DNA molecule is
catalyzed by the enzyme terminal deoxynucleotidyl transferase. The terminal transferase
permits the addition of complementary homopolymer tails (50 to 150 dA or dT long and
about 20 dG or dC long) to 3’ end of plasmid vector and passenger DNA. These tails can
reanneal to form open circular hybrid molecules, which can be ligated in vitro or more
commonly in vivo following transformations to produce functional recombinant molecules.

Selection of recombinants
Making recombinant molecules is a game with very long odds against success. Even when
the bits of DNA have been joined up and inserted into cells, only very few cells out of many
tens of thousands will contain the recombinant molecule and all the technical expertise in
the world is no use whatsoever unless one can find the cell that contain the recombinant
DNA. Techniques for selecting the few valuable cells from the mass of useless ones are
thus of paramount importance.

Directional selection
The phenotypes conferred by the cloned genes on the host are used as markers of
selection. All useful vector molecules carry a selectable genetic marker or have a genetically
selectable property. Plasmid vectors generally possess drug resistance or nutritional
markers and in phage vectors the plaque formation itself is the selectable property.

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Insertional inactivation
The technique depends upon homologous recombination between DNA cloned and the host
genome. If the cloned sequence lacks both promoter and sequences encoding essential
regions of the carboxyl terminus of the protein, recombination with homologous genomic
sequences will cause gene disruption and produce a mutant genotype. On the other hand, if
the cloned fragment contains appropriate transcriptional and translational signals,
homologous recombination will result in synthesis of a functional mRNA transcript, and no
mutant phenotype will be observed.

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 Questions
1. The rDNA technology has provided the means to achieve …………
a). Amplification of cloned genes b). Fractionation of individual DNA
components of complex genomes
c). Potential to create new genetic d). All the above
combinations

2. The other terms that can be used to describe rDNA technology …………
a). Gene manipulation b). Gene cloning
c). Genetic modification d). All the above

3. The essential steps in rDNA technology is …………

a). 4 b). 5
c). 2 d). 7

4. In cDNA synthesis, the eukaryotic ………. is used as a template to generate DNA.

a). mRNA b). tRNA
c). rRNA d). None of the above

5. The enzyme reverse transcriptase is also called as ………

a). RNA dependent DNA polymerase b). DNA dependent DNA polymerase
c). RNA dependent RNA polymerase d). None of the above

6. The chemical synthesis of DNA is by ………

a). Phosphodiester method b). Phosphotriester method
c). Both a and b d). None of the above
.
7. The tool for cutting DNA molecules is/are ………
a). Restriction endonucleases b). Ligases
c). Both a and b d). None of the above

8. The tool for joining DNA molecules is/are ………

a). Restriction endonucleases b). Ligases
c). Both a and b d). None of the above

9. The important tools used in rDNA technology include …………

a). Enzymes for DNA manipulation b). Vectors
c). Expression hosts d).All the above

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 10. The rDNA technology is used for …………
a). Gene cloning b). DNA sequencing
c).Creation of transgenic plants and d).All the above
animals

11. The restriction enzymes are grouped into ………… types

a). 3 b). 5
c). 2 d).None of the above

12. The restriction enzymes used abundantly in rDNA technology is ……..

a). Type I b). Type II
c). Type III d).None of the above

13. The restriction enzyme with star activity is/are ……..

a). EcoRl b). BamHI
c). Sal1 d).All the above

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